ABSTRACT: PROJECT 1: Urine sampling for HPV infection and methylation testing for cervical cancer screening among women living with HIV in Malawi and South Africa Cancer incidence, mortality, and disparities are projected to rise at staggering rates. It is estimated, for example, that cancer will kill one million Africans each year by 2030, with invasive cervical cancer (ICC) accounting for the most cancer deaths in African women. Although global policy seeks to rapidly expand access to human papillomavirus (HPV) vaccination and cervical cancer screening in the coming decade, practical efforts to eliminate cervical cancer in low- and middle-income countries (LMICs) remain limited. This gap leaves generations of women needlessly at risk for ICC, with HIV-positive (HIV+) women particularly vulnerable. The need for cost-effective and scalable screening and treatment strategies to reduce ICC is therefore substantial. There is also an urgent need for quality evidence directly applicable to HIV+ women in LMICs, who face the highest risk of HPV infection, cervical precancer (CIN2/3), and ICC. The central hypothesis of our proposal is that the use of self-collected samples to test for HPV infection has the potential to increase cervical screening coverage in clinical settings where pelvic examinations are not routinely performed. We will validate urine-based HPV testing among HIV+ women attending cervical screening clinics at two strong research sites in Malawi and South Africa. As primary HPV screening is highly sensitive but only moderately specific for CIN2/3 and ICC, we will also validate a novel S5 methylation test for triage of women who screen positive for HPV. In Aim 1, we will enroll 925 HIV+ women and evaluate high-risk HPV testing in urine, self-collected cervicovaginal, and provider-collected cervical samples for the detection of CIN2/3 and ICC (CIN2+). We will report HPV positivity in the 3 sample types and compare the sensitivity, specificity, and positive and negative predictive values (PPV and NPV) for CIN2+ detection between the sample types. In Aim 2, we will evaluate S5 methylation as a triage test for HIV+ women with HPV positive results. For each sample collection method (urine, self, and provider), we will calculate PPV, NPV, sensitivity, and specificity of S5 triage testing for CIN2+. We will also report the number of colposcopy referrals per CIN2+ case detected with and without S5 triage. Our proposed project will be the first to validate urine-based HPV testing among HIV+ women and the first to compare the clinical performance of S5 triage testing using urine, self, and provider-collected samples in an LMIC setting. If successful, our findings will have broad relevance for HIV+ women in both resource-rich and resource-poor regions worldwide, including rural and remote areas of the U.S.